Considerable effort has been expanded in developing ways of regenerating plants from tissue cultures. Several publications are available describing these techniques involving plant regeneration which are listed below for convenience and reference.
Barwale, U. B., Wildholm, J. M. (1989) Whole plant regeneration via organogenesis and somaclonal variation in Glycine species. U.S. Pat. No. 4,857,465; PA0 Brown, D. C., Thrope, T. A. (1986) Plant regeneration and organogenesis. In: Cell culture and somatic cell genetics of plants, I. K. Vasil (ed), Academic Press, pp. 49-65; PA0 Christianson, M. L., Warnick, D. A. (1985) Temporal requirement for phytohormone balance in the control of organogenesis in vitro. Dev. Biol. 112:494-497; PA0 Douglas, G. C. (1990) Manipulation of shoot formation in cultured explants. In: Methods in molecular biology vol. 6, W. Pollard, J. M. Walker (eds). Humana Press, pp. 71-80; PA0 Flick, C. A., Evans, D. A., Sharp, W. R. Organogenesis. In: Handbook of plant cell culture-Techniques and application vol. 4, Evans MacMillan Publishing Co., New York, U.S.A. pp. 370-418; PA0 George, E. F., Sherrington, P. D., (1984) Plant propagation by tissue culture, Exegetics Ltd. U.K., pp. 3; PA0 Goenwald, E. G., Koeleman, A., Wessels, C. J. (1975) Callus Formation and Plant Regeneration from Seed Tissue of Aloe Pretoriensis Pole Evans. Z. Pflanzenphysiol. 75:270-272; PA0 Jackson, J. A., Hobbs, S.L.A. (1990) Rapid multiple shoot production from cotyledonary node explants of pea (Pisum sativum L.). In Vitro Cell Dev Biol 26:835-838; PA0 Skoog, F., Miller, C.O. (1957) Chemical regulation of growth and organ formation in plant tissue cultured in vitro. Soc. Exp. Biol. Symposia 11:118-131; PA0 Skook, F., Miller, C. O. (1957) Chemical regulation of growth and organ formation in plant tissue culture in vitro. Soc. Exp. Biol. Symposia 11:118-131; PA0 Smeltzer, R. H. Cello, L. M. (1982) Tissue culture method for asexual propagation of pine trees and medium for use therewith. U.S. Pat. No. 4,354,327; PA0 Vasil, I. K. (1987) Developing cell and tissue culture system for the improvement of cereal and grass crops. J. Plant Physiol. 128:193-218. PA0 i) culturing an intact, normally healthy plant seed in a suitable medium, said seed being free of contaminants to said medium, PA0 ii) providing in said medium a growth regulator of sufficient concentration to promote a first growth phase where at least one shoot is formed from said seed and subsequently induce a second growth phase in which de novo differentiation in said at least one shoot produces said plurality of regenerants, PA0 iii) continuing culture of said seed during said second growth phase until said regenerants are distinct and well formed. PA0 iv) harvesting said distinct and well formed regenerants; PA0 v) transferring said harvested regenerants to a culture medium which promotes root development; PA0 vi) culturing said transferred regenerants to produce therefrom, seedlings. PA0 i) harvesting said distinct and well formed regenerants; PA0 ii) separating said harvested regenerants; PA0 iii) storing said separated regenerants for subsequent use in developing seedlings therefrom.
There are two pathways of regeneration of plants in tissue culture: Organogenesis and Somatic embryogenesis. Organogenesis is the formation of an organ, a shoot which later develops roots to produce a complete plant and visa versa. Somatic embryogenesis is the formation of somatic embryos which have both shoot and root initials and are capable of developing into whole plants (Brown and Thorpe, 1986).
The process of shoot or embryo regeneration is known to consist of two essential steps: (a) the isolation of an explant from a source seedling and (b) its culture on a nutrient medium supplemented with growth regulators (Christianson and Warnick, 1985, Douglas, 1990; Flick et al. 1983; George and Sherrington, 1984). As is appreciated, explant refers to small pieces of the seedlings cultured for inducing regeneration.
Upon culture, the explant may give rise to adventitious shoots of somatic embryos directly or may produce a mass of undifferentiated cells referred to as callus. The callus then can be made to differentiate into shoots or somatic embryos (Brown and Thorpe, 1986; George and Sherrington, 1984) by culturing on media supplemented with growth regulators.
The discovery of the principle of regeneration in tissue cultures was made in 1957 by Skoog and Miller. It was suggested that all types of cell growth and differentiation from tissue/callus cultures is controlled by the balance of auxins and cytokinins in the medium. The theory has been found to be true in hundreds of studies subsequently conducted by many researches (Flick et al. 1983; George and Sherrington, 1984).
Consequently, it is now well established that an explant can be made to differentiate into shoots or somatic embryos by minor variations in hormonal balance and source of explants. For instance in pea, there are several reports describing somatic embryogenesis and shoot regeneration (Tetu, T., Sanywan, R. S., and Sangwan-Neyyeal, B. S., Direct Somatic Embryogenesis and Organogenesis in Cultured Immature Zygotic Embryos of Pisum sativum L., J. Plant Physiol. 137:102-109, 1990).
In many instances for example geranium (Wilson, Qureshi and Saxena, 1990, Unpublished), lentil, beans, carrot (Malik and Saxena unpublished) somatic embryos are discernable for a very brief period and the end products are shoots. Since the fate of cell to produce somatic embryo or shoot is governed by hormonal balance and this said balance is influenced by a variety of factors, such as type of explant, culture medium, temperature and light, the possibility of the development of either a shoot or an embryo independently or simultaneously is very strong. Simultaneous occurrence of shoot and somatic embryos was seen in many cultures in our experiments (e.g., in Arachis hypogea, Phaseolus coccenius, P. wrightii, Pelargonium hortorun). For the purpose of multiple plant regeneration, the subject of this invention, it is not important if the end products, the shoots, are developed from structures which started as a shoot or as an embryo, but later developed into shoots. Therefore, the term regenerants shall be used hereafter to describe the regenerated plants. Regenerants (Ro) is a commonly used term to describe the regenerated plants of tissue culture original [Maclean, P. and Grafton, K. F., Regeneration f Dry Bean (Phaseolus volgaris L.) via Organogenesis, Plant Science, 6:117 (1989); Franklin, C. I., Trieu, T. N., Gonzales, R. A., Dixon, R. A., Plant Regeneration from Seedling Explant of Green Beans (Phaseolus vulgaris L.) via Organogenesis, Plant Cell Tissue and Organ Culture 24:199-206 (1991)] irrespective of their origin as a shoot or an embryo.
The success in regenerating plants from a wide variety of species via organogenesis or somatic embryogenesis in plant tissue cultures by manipulating quantitative interaction of phytohormones has resulted in a definite pattern of experimental approach to achieve regeneration. The procedure involves preparation of explants from seedlings raised from a seed (stage I), culture of explants (stage II), and incubation to allow growth and differentiation (stage III) as shown in Flow Chart 1. ##STR1##
There seems to be a consensus that the success in inducing regeneration depends upon the choice of the explant and the nutritional and physical milieus of explant culture (Brown and Thorpe, 1986). Thus, a great deal of research has been directed towards the optimization of physiological conditions of the source seedlings, selection and culture of explants, and the phytohormones used to initiate tissue cultures (stages I, II, and III in Chart 1). The see (stage 0 in Chart 1) has also been used as an explant but prior to culture it was cut into smaller segments (Groenwald et al., 1975).
However, whole embryos separated mechanically from the seed have been used quite successfully in many plants including cereals and legumes (U.S. Pat. No. 4,857,465 and Vasil, 1987). For example, Barwale et al. (1989) isolated young embryos to develop regenerating callus cultures of soybean (U.S. Pat. No. 4,857,465).
In one previous development, Smeltzer et al. (1982) used nuclear tissue from seeds to obtain multiple shoots (U.S. Pat. No. 4,354,327). Smeltzer et al. provided a method for recovering multiple shoot buds capable of forming plants. As many as ten regenerants from one seed, was described by Smeltzer et al. However, the data given therein showed only percentages of seeds giving buds in response to a particular treatment; it was not clear if all treatments produced two or in between two and ten buds per seed. In addition, a maximum of four germinating seeds were used to estimate the percent seed with buds. The procedure of Smeltzer et al. essentially comprises stimulation of seed germination followed by surface serialization and culture in a nutrient medium containing BAP and abscisic acid (ABA). The method of stimulation of seed involved making a rook end or micropylar cut into the endosperm of the seed and then immersing the seed in a 1% hydrogen peroxide solution for one week. Thereafter, seed coat was removed with a thumbnail and isolated nucellar tissue was surface sterilized before inserting the protruding root radicle into culture medium. Such removal of seed coat following a cut into the endosperm results in injury to the seed tissue, but for purposes of their technique such injury is not critical.
Our discovery involves the use of whole, completely intact seeds where, during culture thereof, the intactness of the seedling derived from the cultured seed is maintained. The term intack implies that the seed is physically uninjured, ungerminated and viable for purposes of culture in developing a seeding bearing differentiated regenerants.